PROJECT SUMMARY Biomolecules with the capacity for diverse molecular recognition are essential reagents in biomedical research and have been recently developed into uniquely efficacious anti-cancer treatments. In particular, antibody mimetic proteins (AMPs) that function both inside and outside cells have been useful for controlling target protein localization, blocking functional interfaces on target proteins, and mediating cytotoxic effects to target-expressing cancer cells. However, a crucial missing feature of existing AMPs is the ability for target binding to be controlled remotely, in real time, and in defined points in space. Optical control of AMP binding would be immensely and widely useful throughout the biosciences by enabling protein sequestration or functional blockade in living cells to be controlled exquisitely in time and space. In particular, photocontrollable AMPs would be powerful reagents for controlling the activity endogenous proteins with spatiotemporal specificity, especially proteins for which no specific small molecule inhibitors exist. Photocontrollable AMPs could also be used to direct immune cells to cancer cells at specific times or at specific locations in the body. We propose to develop the ability to optically control AMP-target interactions using protein domains that can be photoswitched from dimeric to monomeric states by cyan light. We propose that, by inserting two copies of these photoswitchable domains to constant (non-variable) regions in AMPs, we can create AMPs that do not interact with their targets in the dark due to binding site occlusion, but that do bind to targets after cyan illumination. All possible rational designs will be explored, involving using scaffolds of the DARPin and repebody families unmodified or after topological modification, and inserting photodissociable domains at termini and/or interior loops. Specifically, we will (1) engineer photoswitchable DARPins and repebodies by inserting photoswitchable domains into loops and termini of existing DARPins, (2) Engineer photoswitchable DARPins by redesigning the framework to introduce new sites for photodissociable domain insertion while improving protein stability, and (3) Test the ability of photoswitchable DARPins and repebodies to perform light-controlled inhibition, relocalization, or degradation of proteins inside cells and recruitment of immune cells to cells expressing cancer antigens. If successful, this work will establish photoswitchable AMPs as a new type of reagent with exceptionally broad applicability in biology. By allowing binding to nearly any endogenous protein to be remotely control with high spatiotemporal precision, photoswitchable AMPs can give researchers the ability to investigate dynamic processes in living cells in unprecedented detail, and give clinicians the ability to recruit the immune system to specific targets at specific times and in specific regions of the body.